Stroke is the major neurological illness and recombinant tissue type plasminogen activator (rtPA) is the only approved treatment. Use of rtPA is limited to three hours after stroke because of the risk of intracerebral hemorrhage. Toxicity of rtPA occurs when the blood-brain barrier (BBB) is opened. Prevention of the BBB damage expands the therapeutic window for the use of rtPA in stroke. Matrix metalloproteinases (MMPs) are increased in brain after hypoxic/ischemic injury: MMPs disrupt the blood-brain barrier (BBB), causing edema, hemorrhage, and cell death. Others and the PI have shown that treatment with MMP inhibitors (MMPIs) prevent the early opening of the BBB and reduce death and hemorrhage in animal models of stroke. We propose to use in vitro screening of a number of novel MMPIs that have been patented by the Co-PI, and to test the most promising ones in animal models of stroke. The goal is to identify MMPIs that could be used to obtain an IND for translational clinical trials in the U01 program. Our goal is to rationally design, synthesize, and test MMPIs that could be used in treatment trials of MMPIs in stroke. These compounds block the MMPs involved in opening the BBB in the early phases of injury and may expand the therapeutic window for rtPA treatment. We plan to use in vitro methods to screen a large number of possible drugs. Those with the ability to block the BBB will be tested in vivo with isotopes. When several candidate drugs are identified, MRI will be used to simulate the clinical conditions with noninvasive studies of the early and subacute events, allowing for and behavioral testing in the same animals to assure that there is no interference with long- term recovery. Specific aims: 1) To develop and test in vitro the efficacy of the potential therapeutic agents that block the MMPs, using enzyme inhibition kinetic assays and 3-dimensional human brain microvascular endothelial and astrocyte or glia cell co-culture systems. 2) To determine the ability and efficacy of a new generation of MMP inhibitors (MMPIs) recently developed and patented by Sang and colleagues to block the early opening of the BBB as assessed by isotopes and MRI in animal models. 3) To select promising agents that block the BBB and to measure the effect on stroke infarct size at 48 hrs when the damage is greatest. 4) To further select the most efficacious agents for testing long-term effects on behavior, recovery, and survival in animal models. Significance: MMPIs protect the BBB and have been shown to extend the therapeutic window for use of rtPA in animal studies. This proposal will combine the resources of experts in MMPI drug design and measurements of BBB damage in vivo and will utilize start-of-art MRI technology to identify the most efficacious novel MMPIs for testing during the early phases of stroke. The goal of this two-year drug discovery project is to identify one or more lead compounds for later testing under the full U01 mechanism. PUBLIC HEALTH RELEVANCE: Stroke is the leading neurological cause of death and disability. Treatment with rtPA has been proven beneficial, but a constricted therapeutic window limits its use. Animal studies have shown that matrix metalloproteinase inhibitors (MMPIs), which reduce damage to the blood-brain barrier (BBB), attenuate the toxicity of rtPA and expand the therapeutic window. This project combines the expertise of a MMPI synthesis laboratory and a group with expertise in animal models of stroke. The goal is to identify several lead compounds that can be used in clinical trials. Novel MMPIs will be screened in cell cultures and cell-based models of the BBB. Promising agents will be tested in animals with isotopes. Finally, those agents that pass these initial studies will be tested with MRI measurements of BBB and infarct size with the addition of behavioral studies to show long-term recovery. The overall goal is to identify lead compounds that are patentable and can be used to obtain IND approval. Promising compounds would be useful for U01- funded clinical trials.